Disease resistance in Vitis

ABSTRACT

Disclosed are methods for producing grape plants having resistance to bunch rot disease or  Botrytis  or both. Also disclosed are grape plants having resistance to bunch rot disease or  Botrytis  or both, wherein the plants express a cecropin B peptide Shiva-1.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of Ser. No. 09/876,202, filed Jun. 6,2001, now abandoned; which is a continuation of Ser. No. 09/511, 606,filed Feb. 23, 2000, now abandoned; which is a continuation of Ser. No.09/452,577, filed Dec. 01, 1999, now abandoned; which is a continuationof Ser. No. 08/878,750, filed Jun. 19, 1997, now U.S. Pat. No.6,232,528, which claims priority from provisional application60/020,569, filed Jun. 26, 1996.

BACKGROUND OF THE INVENTION

This application relates to disease resistance in Vitis.

Grape (Vitis spp.) is a deciduous temperate fruit crop of ancientorigin. Grape production (65×10⁶ metic tons) exceeds that of any othertemperate fruit crop and ranks after Citrus and banana among all fruitcrops worldwide (FAO Production Yearbook, 1990). Grape surpasses allother fruit crops in value due to its multiple uses for fresh fruit,juice, jelly, raisins, and wine. For example, in the United States,seedless grapes represent about 80% and 98% of the total table andraisin grape production, respectively (In: 1994–95: The Distribution andPer Capita Consumption of California Table Grapes By Major Varieties inthe United States and Canada, California Table Grape Commission, Fresno,Calif. 1995). Only a few seedless cultivars make up this production, ofwhich ‘Thompson Seedless’ is the most important. This cultivar accountsfor the most production of any single grape variety in the UnitedStates. In 1992, ‘Thompson Seedless’ was grown on 263,621 acres inCalifornia (In: California Grape Acreage, California AgriculturalStatistics Service, Sacramento, Calif., 1993). Thirty-five percent ofthe table grape production in California in 1994 was ‘Thompson Seedless’(23,244,683 boxes, 10 kg/box). In 1993, 97% of the grapes grown forraisin production was ‘Thompson Seedless’ (In: Raisin CommitteeMarketing Policy 1994–95, Raisin Administrative Committee, Fresno,Calif., 1994).

Although Vitis spp. is generally considered to have desirable fruitquality, it is susceptible to many pests and diseases, includinganthracnose, black rot, botrytis bunch rot, crown gall, downy mildew,eutypa dieback, various nematodes, phomopsis cane and leaf spot,phylloxera, Pierce's disease, and powdery mildew. Hybridization withresistant species has been the only method available to produceresistant cultivars (Galet and Morton, In: Compendium of Grape Diseases,R. C. Pearson and A. C. Goheen, eds., APS Press, St. Paul, 1990, pp.2–3). While improving grape is possible by conventional breeding, it isdifficult and time consuming due to the two- to three-year generationcycle, the long period of time required for reliable progeny testing andselection, and inbreeding depression that prohibits selfing (Gray andMeredith, In: Biotechnology of Perennial Fruit Crops, F. A. Hammerschlagand R. E. Litz, eds., C.A.B. Intl., Wallingford, U.K. 1992). Thesecharacteristics make introgression of desirable traits into existinggrape cultivars difficult if not impossible to achieve in an individualbreeder's lifetime. Thus, the alternative, and potentially lesstime-consuming, approach of using gene transfer to insert desirablegenes is one approach for improving grapevine cultivars, evenconsidering the time necessary for field testing transgenic lines. Theability to improve the disease or pest resistance or both of a majorgrape cultivar (e.g., ‘Thompson Seedless’) offers the possibility ofimproving a large portion of the grape production in a relatively shorttime, assuming that cultivar integrity would not be compromised by thetransgene or the insertion event. Such a change could also reducepesticide use for a significant portion of grape production.

SUMMARY OF THE INVENTION

In one aspect, the invention features a method for producing atransgenic plant of the genus Vitis having resistance to a plantpathogen. The method, in general, includes the step of transforming aplant cell with a nucleic acid which expresses a lytic peptide, wherethe expression of such a lytic peptide provides resistance to a plantpathogen. In preferred embodiments, the method further includespropagating a grape plant from the transformed plant cell. In otherpreferred embodiments, the method involves transforming a plant cellthat is a part of a somatic embryo and propagating or regenerating atransgenic grape plant from the transformed somatic embryo. Expressionof the lytic peptide confers disease resistance or tolerance or both tograpevine pathogens and pests including, without limitation, bacterial,fungal, and viral pathogens.

In general, Vitis is transformed by introducing into a plant cell orsomatic or zygotic embryos a nucleic acid that includes a lytic peptideby using A. tumefaciens, microprojectile bombardment, or any combinationof these methods (for example, by bombarding the plant cell withmicroprojectiles, followed by infecting the bombarded cells withAgrobacterium tumefaciens including a nucleic acid which expresses thelytic peptide).

In preferred embodiments, the method of the invention involves the useof the lytic peptides Shiva-1 or cecropin B or both.

The methods of the invention are useful for providing disease resistanceor tolerance or both to a variety of grape plants (for example, Vitisspp., Vitis spp. hybrids, and all members of the subgenera Euvitis andMuscadinia), including scion or rootstock cultivars. Exemplary scioncultivars include, without limitation, those which are referred to astable or raisin grapes and those used in wine production such asCabernet Franc, Cabernet Sauvignon, Chardonnay (e.g., CH 01, CH 02, CHDijon), Merlot, Pinot Noir (PN, PN Dijon), Semillon, White Riesling,Lambrusco, Thompson Seedless, Autumn Seedless, Niagrara Seedless, andSeval Blanc. Rootstock cultivars that are useful in the inventioninclude, without limitation, Vitis rupestris Constantia, Vitis rupestrisSt. George, Vitis california, Vitis girdiana, Vitis rotundifolia, Vitisrotundifolia Carlos, Richter 110 (Vitis berlandieri×rupestris), 101–14Millarder et de Grasset (Vitis riparia×rupestris), Teleki 5C (Vitisberlandieri×riparia), 3309 Courderc (Vitis riparia×rupestris), RipariaGloire de Montpellier (Vitis riparia), 5BB Teleki (selection Kober,Vitis berlandieri×riparia), SO₄ (Vitis berlandieri×rupestris), 41BMillardet (Vitis vinifera×berlandieri), and 039-16 (Vitisvinifera×Muscadinia).

In another aspect, the invention features a transgenic plant or plantcell of the genus Vitis transformed with a nucleic acid which expressesa lytic peptide, wherein expression of the lytic peptide providesresistance to a plant pathogen. In preferred embodiments, the transgenicgrapevine or cell with the nucleic acid includes an expression vector.Preferably, the transgenic grapevine or cell is Vitis vinifera ‘ThompsonSeedless’ and the expression of the lytic peptide provides resistance tothe bacterium Xylella fastidiosa, the causative agent of Pierce'sDisease. In other preferred embodiments, the transgenic grapevine is asomatic embryo, a scion, or a rootstock.

In still another aspect, the invention features a method of transformingVitis with a nucleic acid which expresses a tomato ringspot virus coatprotein (TomRSV-CP) gene, where the expression of such a coat proteingene provides resistance to a plant pathogen.

In still another aspect, the invention features a method of transformingVitis with a nucleic acid which expresses a TomRSV-CP gene and a lyticpeptide gene, where the expression of such genes in a grapevine providesresistance to a plant pathogen.

The invention also features scions, rootstocks, somatic or zygoticembryos, cells, or seeds that are produced from any of the transgenicgrape plants described herein.

By “lytic peptide” is meant a gene encoding a polypeptide capable oflysing a cell. Exemplary lytic peptides include, without limitation,apidaceins, attacins, cercropins (e.g., cercropin B), caerulins,bombinins, lysozyme, magainins, melittins, sapecin, sarcotoxins, andxenopsins.

By “peptide” is meant any chain of amino acids, regardless of length orpost-translational modification (for example, glycosylation orphosphorylation).

By “positioned for expression” is meant that the DNA molecule ispositioned adjacent to a DNA sequence which directs transcription andtranslation of the sequence (i.e., facilitates the production of, forexample, a lytic peptide).

By “operably linked” is meant that a gene and a regulatory sequence(s)are connected in such a way as to permit gene expression when theappropriate molecules (for example, transcriptional activator proteins)are bound to the regulatory sequence(s).

By “plant cell” is meant any self-propagating cell bounded by asemi-permeable membrane and containing a plastid. Such a cell alsorequires a cell wall if further propagation is desired. A plant cell, asused herein, is obtained from, without limitation, seeds, suspensioncultures, embryos, meristematic regions, callus tissue, leaves, roots,shoots, somatic and zygotic embryos, as well as any part of areproductive or vegetative tissue or organ.

By “transgenic” is meant any cell which includes a DNA sequence which isinserted by artifice into a cell and becomes part of the genome of theorganism which develops from that cell. As used herein, the transgenicorganisms are generally transgenic grapevines and the DNA (transgene) isinserted by artifice into the nuclear or plastidic genome. A transgenicgrapevine according to the invention contains at least one lytic peptideor TomRSV-CP or both.

By “transgene” is meant any piece of DNA which is inserted by artificeinto a cell, and becomes part of the genome of the organism whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism.

As discussed above, we have discovered that the expression of a lyticpeptide provides grapevines with resistance against disease caused byplant pathogens and pests. Accordingly, the invention provides a numberof important advances and advantages for viticulturists. For example, bydemonstrating that the lytic peptide Shiva-1 is effective againstXyellela fastidiosa, the invention facilitates an effective andeconomical means for protection against Pierce's Disease. Suchprotection reduces or minimizes the need for traditional chemicalpractices that are typically used by viticulturists for controlling thespread of plant pathogens and providing protection againstdisease-causing pathogens in vineyards. In addition, because grapeplants expressing one or more lytic peptide gene(s) described herein areless vulnerable to pathogens and their diseases, the invention furtherprovides for increased production efficiency, as well as forimprovements in quality, color, flavor, and yield of grapes.Furthermore, because the invention reduces the necessity for chemicalprotection against plant pathogens, the invention benefits theenvironment where the crops are grown. In addition, the expression of alytic peptide gene or TomRSV-CP or both in a grapevine providesresistance to plant pathogens and can be used to protect grapevines frompathogen infestation that reduces productivity and viability. Themethods of the invention are useful for producing grapevines havingresistance to diseases including, without limitation, Pierce's disease,crown gall, bunch rot, downy and powdery mildews, and viral diseasescaused by arabis mosaic virus, grapevine fanleaf virus, tomato ringspotvirus, grapevine leafroll associated virus.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DETAILED DESCRIPTION

The drawings will first be described.

DRAWINGS

FIG. 1A shows the partial map of the T-DNA region of pBRS1.

FIG. 1B shows the partial map of the T-DNA region of pGA482GG/cpTomRSV.

FIG. 2 is a photograph showing the results of PCR amplified TomRSV-CPand Shiva-1 fragments from transgenic ‘Thompson Seedless’ grape plants.PCR analysis using TomRSV-CP primers are as follows: pGA482GGtransformant (without the TomRSV-CP gene); lane 2, transformant 3-2;lane 3, transformant 3-3; lane 4, transformant 3S-2; lane 5,transformant 3S-6; lane 6, transformant 3SB-X. PCR analysis usingShiva-1 primers are as follows: lane 7, untransformed ‘ThompsonSeedless’ plant; lane 8, transformant 4-3; lane 9, transformant 4S-2.Transgenic plants 3-2, 3-3, and 4-3, were obtained from A. tumefaciensinfection alone. Plants 3S-2, 3S-3, 3SB-X, and 4S-2 were obtained fromA. tumefaciens infection after microprojectile bombardment.

FIG. 3 is a photograph showing the results of a Southern analysis oftransgenic ‘Thompson Seedless’ grape plants. DNA extracted fromTomRSV-CP transformants that was digested with EcoRI and probed with aNOS/NPTII fragment is shown in lanes 1 through 9. Lane 1, pGA482GGtransformant (control without the TomRSV-CP gene); lane 2, transformant3-2; lane 3, transformant 3-3 from tissue culture; lane 4, transformant3-3 from greenhouse leaves (DNA runs slower on gel); lane 5,transformant 3S-2; lane 6, transformant 3S-3; lane 7, transformant3SB-X; lane 8, untransformed control ‘Thompson Seedless’; lane 9,pGA482GG/cpTomRSV plasmid. Shiva-1 transformants digested with BamHI andprobed with a NOS/NPTII fragment are shown in lanes a–c. Lane a,transformant 4-3; lane b, transformant 4S-2; lane c, untransformedcontrol ‘Thompson Seedless’. Transgenic plants 3-2, 3-3, and 4-3 wereobtained from A. tumefaciens infection alone. Plants 3S-2, 3S-3, 3SB-X,and 4S-2 were obtained from A. tumefaciens infection aftermicroprojectile bombardment.

FIG. 4 is a photograph showing a transgenic ‘Thompson Seedless’ grapeplant four months after transfer to the greenhouse.

A description for the production of disease resistant transgenic Vitisnow follows. Transgenic grape plants expressing either lytic peptide orTomRSV coat protein genes were regenerated from somatic embryos derivedfrom leaves of in vitro-grown plants of ‘Thompson Seedless’ grape (Vitisvinifera L.) plants. Somatic embryos were either exposed directly toengineered A. tumefaciens or they were bombarded twice with 1-μm goldparticles and then exposed to A. tumefaciens. Somatic embryos weretransformed with either the lytic peptide Shiva-1 gene or the tomatoringspot virus coat protein (TomRSV-CP) gene. Integration of the foreigngenes into these grapevines was verified by growth in the presence ofkanamycin (kan), positive β-glucuronidase (GUS) and polymerasechain-reaction (PCR) assays, and Southern analysis. Resistance toPierce's disease in transgenic plants expressing a lytic peptide wasalso examined. These examples are provided for the purpose ofillustrating the invention, and should not be construed as limiting.

Plant Materials and Culture

Leaves from ‘Thompson Seedless’ in vitro cultures were used to producesomatic embryos following the method of Stamp et al. (J. Amer. Hort.Sci. 115:1038–1042, 1990). Expanding leaves (approximately 0.5 cm long)excised from in vitro-grown shoots were cultured on a modified Nitschand Nitsch (Science 163:85–87, 1969) (NN) medium containing 5 μM of2,4-D, 1 μM of BA, 60 grams/liter of sucrose, 2 grams/liter of activatedcharcoal, and 7 grams/liter of agar, pH 5.7. After a three- totwelve-week culture period, somatic embryos formed. These weretransferred to a modified Murashige and Skoog (Plant Physiol.15:473–497, 1962) (MS) medium containing 120 grams/liter of sucrose, 2grams/liter of activated charcoal, and 7 grams/liter of agar, pH 5.7.After three years of continual culture on the modified MS medium withtransfers each four to six weeks, somatic embryos were transferred toEmershad and Ramming proliferation (ERP) medium (Emershad and Ramming,Plant Cell Rpt. 14:6–12, 1994) for several transfers and then exposed totransformation treatments.

Agrobacterium Strain and Plasmid Descriptions

For the transformation treatments described below, A. tumefaciensstrains were EHA101 and EHA105 (Hood et al., J. Bacterial.168:1283–1290, 1986) containing plasmid pGA482GG/cpTomRSV (Slightom,Gene 100:252–255, 1991; Slightom et al., In: Plant Mol. Biol. Man., S.B. Gelivn, R. A. Schilperoot, and D. P. S. Verma, eds., Kluwer,Dordrecht, The Netherlands) or pBPRS1, respectively, were used (FIGS.1A–1B). Both plasmids contained chimeric gusA (β-glucuronidase (GUS))and kanamycin (Kan) (neomycin phosphotransferase II (NPT II)) genes.Plasmid pGA482GG/cpTomRSV contained the tomato ringsport virus coatprotein (TomRSV-CP) gene and pBRPS contained the Shiva-1 lytic peptidegene (Destefano-Beltran et al., In: The Molecular and Cellular Biologyof the Potato, M. Vayada and W. Parks, eds., C.A.B. Int'l Wallingford,U.K.; Jaynes et al., Acta Hort. 336:33–39, 1993).

Cocultivation and Selection

Putative A. tumefaciens transformants were cocultivated and selected asdescribed by Scorza et al. (Plant Cell Rpt. 14:589–592, 1995). Briefly,A. tumefaciens cultures were grown overnight at 28° C. in LB mediumcontaining selective antibiotics for each plasmid. These cultures werecentrifuged (5,000 ×g, 10 minutes) and resuspended in a mediumconsisting of MS salts containing 20 grams/liter of sucrose, 100 μM ofacetosyringone, and 1.0 μM of betaine phosphate. The cultures were thenshaken for about six hours at 20° C. before use in the transformationtreatments that are described below.

Transformation

Somatic embryos were either bombarded with gold microprojectiles andthen exposed to A. tumefaciens as described by Scorza et al. (J. Amer.Soc. Hort. Sci. 119:1091–1098, 1994) or they were exposed to A.tumefaciens without prior bombardment as follows. Microprojectilebombardment was accomplished using the Biolistic PDS-1000/He device(Bio-Rad Laboratories). A total of 700 somatic embryos were separatedinto groups of 100. Each group was placed onto a 25-mm polycarbonatemembrane in the center of a 100-mm petri plate containing ERP mediumtwenty-four hours before bombardment. Somatic embryos were shot with1.0-μm diameter gold particles following the general procedures ofSanford et al. (Meth. Enzmol. 217:483–509, 1991) using the parametersdescribed by Scorza et al. (Plant Cell Rpt. 14:589–592, 1995). Allplates were bombarded twice. Within two hours of bombardment, embryoswere cocultivated with A. tumefaciens. After bombardment, somaticembryos were immersed in the resuspended A. tumefaciens culture that wasprepared as described above. After fifteen to twenty minutes, the A.tumefaciens culture medium was removed and somatic embryos were placedonto cocultivation medium (ERP medium containing 100 μm acetosyringone).Somatic embryos were cocultivated for two days and then washed withliquid ERP medium (without charcoal) containing 300 μg/ml of cefotaximeand 200 μg/ml of carbenicillin. Somatic embryos were then plated onagar-solidified ERP medium (0.75% agar) with the above-mentionedselective antibiotics. All somatic embryo cultures were allowed toproliferate for two passages (3 weeks each) before being placed ontoselection medium. Selection was carried out on ERP medium containing theabove specified amounts of cefotaxime and carbenicillin, and 40 μg/ml ofkanamycin.

In a second series of transformation experiments, an additional 700somatic embryos were exposed to A. tumefaciens without prior bombardmentaccording to the methods described above.

After cocultivation and selection on ERP medium, putatively transformedembryos were induced to germinate and root on woody plant medium (Lloydand McCown, Proc. Intl. Plant Prop. Soc. 30:421–427, 1981) containing 15grams/liter of sucrose, 1 μM of BA, 3 grams/liter of agar, pH 6.0following the protocol of Emershad and Ramming (Plant Cell Rpt. 14:6–12,1994).

Transformation Confirmation

Transformed somatic embryos and shoots produced after somatic embryogermination were assayed by growth on kanamycin-containing medium andthrough a histological GUS assay (Jefferson, Plant Mol. Biol. Rpt.5:387–405, 1987). Leaf samples of the plants surviving kanamycinselection were observed to produce the characteristic blue GUS positivereaction, indicating the presence and activity of the GUS gene in theseplants. Leaves from untransformed control plants showed no bluestaining.

Leaves sampled from plants growing in vitro were also cultured for oneweek in liquid LB medium to assay for the presence of contaminating A.tumefaciens. Excised leaves from putative transformants cultured inliquid LB medium were negative for the presence of contaminating A.tumefaciens.

After rooting and transfer to the greenhouse, transformed plants weresubjected to PCR and Southern analysis. PCR amplification was conductedon DNA isolated from leaves of putatively transformed grape plants.Specific oligonucleotide primers from TomRSV-CP and Shiva-1 genesequences were used to identify the presence of these genes in DNA fromthe different clones. For the TomRSV-CP gene, these sequences were the5′ primer 5′-GGTTCAGGGCGGGTCCTGGAAG-3′ (SEQ ID NO:1) and 3′ primer5′-GTAAAAGCTAATTAAGAGGCCACC-3′ (SEQ ID NO:2); for Shiva-1 gene, thesequences were the 5′ primer 5′-ATCAAACAGGGTATCCTGCG-3′ (SEQ ID NO:3)and 3′ primer 5′-TTCCCACCAACGCTGATC-3′ (SEQ ID NO:4). PCR reactions wererun using the GeneAmp kit components (Perkin Elmer, Norwalk, Conn.)using the following parameters: 1 minute at 94° C., 1.5 minutes at 65°C., and 2 minutes at 72° C. The first cycle used an additional 3 minutesmelt at 95° C. and the last five cycles had a 4 minute extension timeperiod at 72° C. After thirty-five amplification cycles, the PCRproducts were analyzed by agarose gel electrophoresis and stained withethidium bromide. PCR analysis using TomRSV-CP and Shiva-1 primersindicated that the thirteen plants that survived kanamycin selectionafter being exposed to TomRSV-CP or Shiva-1 transformation treatmentscontained the predicted gene sequences (FIG. 2).

In addition, Southern analysis was used to demonstrate the incorporationof the foreign genes into the grape genome. Southern analysis wascarried out using a PCR-generated 1.1-kb NOS/NPTII probe. Digestion withEcoRI was then used to test for unique insertion events that wouldinclude segments of grape DNA in pGA482GG/cpTomRSV transformants. BamHIrestriction digestion was used for the pBPRS1 (Shiva-1) transformants.Extraction of DNA from transformants followed the procedures of Callahanet al. (Plant Physiol. 100:482–488, 1992). Conditions for Southernanalysis were described by Scorza et al. (In Vitro Cell Dev. Biol.26:829–834, 1990). The NOS/NPTII probe was radioactively labeledaccording to standard methods using random primers according to theinstructions with the BioRad Random Primer DNA Labeling Kit (BioRad,Hercules, Calif.).

While Southern analysis directly showed only the incorporation of theNPTII gene into the genomes of the assayed grape plants, the closelinkage of the TomRSV-CP or the Shiva-1 genes to the NPTII gene coupledwith the positive PCR assays for the presence of these genes leads tothe conclusion that these plants also contained the TomRSV or Shiva-1genes. This analysis also indicated that most TomRSV-CP transformantscontained multiple copies of the gene insert. Shiva-1 transformants,however, appeared to contain a single insert. Plasmid pGA482GG was usedfor transferring the TomRSV-CP gene. Previous work using plasmidpGA482GG for transforming grape and other species suggested thatmultiple copy transformants are common (Scorza et al., J. Amer. Soc.Hort. Sci. 119:1091–1098, 1994; Scorza et al., Plant Cell Rpt.14:589–592, 1995).

Previous work examined the use of microprojectile bombardment with A.tumefaciens to produce transgenic grape plants. Here we used bothmicroprojectile bombardment and A. tumefaciens infection. Althoughmicroprojectile bombardment before A. tumefaciens infection improved theyield of transformants, the numbers of transformants obtained in thisstudy were too low to be compared with infection with A. tumefaciensinfection alone. It is apparent, however, that both microprojectilebombardment followed by exposure to A. tumefaciens and A. tumefaciensinfection alone are effective for transforming grape somatic embryos.The overall transformation rate in terms of transgenic plants producedper somatic embryo treated was about 1% (Table 1).

TABLE 1 Somatic Putative Treatment Embryos Transformants TransformationAgrobacterium tumefaciens alone Control plasmid 100 1 1.00 TomRSV-CP 3002 0.67 Shiva-1 300 2 0.67 Particle bombardment plus A. tumefaciensControl plasmid 100 1 1.00 TomRSV-CP 300 7 2.30 Shiva-1 300 2 0.67

The results described here differ from our previous report in that wenow report transforming grape from somatic embryos derived from leaves,while previously we reported producing transgenic plants from somaticembryos derived from zygotic embryos. The genes transferred include aviral coat protein gene and a lytic peptide gene. To date there havebeen few reports of transgenic grapevine production, and our resultsdocument the successful transformation of a major Vitis vinifera scioncultivar.

Disease Resistance

Resistance to Pierce's Disease (PD) has been evaluated in transgenic‘Thompson Seedless’ grapevines expressing the lytic peptide Shiva-1. PDis a fatal disease of grapevine known throughout the world. PD killsgrapevines by blocking the plant's water-transporting tissue, the xylem.The disease is caused by the bacterium, Xyllella fastidiosa, and isspread by a leafhopper, the blue-green sharpshooter that feeds on thexylem fluid of grape. The sharpshooter transmits the bacteria from vineto vine. As the bacteria multiply inside the plant, they plug the xylemvessels, inhibiting water and nutrient transport throughout the plant.Infected vines die for reasons related to water uptake. The symptoms ofPD therefore resemble those of water stress and include the drying,marginal burning, or scorching of leaves due to initial clogging of finevessel elements, and eventual dieback of the vine due to total occlusionof the vessels in the trunk. Other symptoms include the shriveling anddying of fruit clusters.

Three transgenic grapevines expressing the Shiva-1 construct have beenevaluated for resistance to X. fastidiosa. These included anon-transformed control; a transformed grapevine containing one Shiva-1insert (designated clone B); and a transgenic grapevine containing fourShiva-1 inserts (designated clone A). Each of these plants werevegetatively propagated and then inoculated with X. fastidiosa accordingto the methods described by Hopkins (Phytopathology 75:713–717, 1985).While replicate plants of all three clones eventually succumbed to PD,clone A was observed to exhibit milder PD symptomology, which did notinclude the typical signs of marginal leaf burn when compared to thenon-transformed control plant. Instead the leaves of clone A slowlybecame chlorotic, without signs of marginal burn. A second series ofinoculations were performed with the same results. In addition, thegrowth of bacteria in the transgenic clones was evaluated and comparedto the non-transformed control plant. Although bacteria were eventuallyfound in the leaves of both transgenic and non-transformed plants, thespread of bacteria was slower in clone A. Our results therefore indicatethat transgenic grapevine expressing the lytic peptide Shiva-1 areeffective at inhibiting PD.

The methods of the invention are also useful for providing resistance toother grapevine diseases. Transgenic grapevines expressing a transgenecontaining a lytic peptide (e.g., Shiva-1 or cecropin B) or TomRSV-CP orboth are operably linked to a constitutive promoter or to a controllablepromoter such as a tissue-specific promoter, cell-type specificpromoter, or to a promoter that is induced by an external signal oragent such as a pathogen- or wound-inducible control element, thuslimiting the temporal or tissue expression or both. Such transgenes mayalso be expressed in roots, leaves, or fruits, or at a site of agrapevine that is susceptible to pathogen penetration and infection. Forexample, a lytic peptide gene may be engineered for constitutive lowlevel expression in xylem-tissue expression and then transformed into aVitis host plant. To achieve pathogen resistance or disease resistanceor both, it is important to express the transgene at an effective level.Evaluation of the level of pathogen protection conferred to a plant byexpression of such a transgene is determined according to conventionalmethods and assays as described herein.

In one working example, expression of a lytic peptide (e.g., Shiva-1 orcecropin B) is used to control bacterial infection, for example, tocontrol Agrobacterium, the causative agent of crown gall disease.Specifically, the Shiva-1 expression vector described herein or a plantexpression vector containing the cecropin B gene is used to transformsomatic embryos according to the methods described above. To assessresistance to Agrobacterium infection and crown gall formation,transformed plants and appropriate controls are grown, and the sternsare inoculated with a suspension of Agrobacterium according to standardmethods. Transformed grape plants are subsequently incubated in a growthchamber, and the inoculated stems are analyzed for signs of resistanceto crown gall formation according to standard methods. For example, thenumber of galls per inoculation are recorded and evaluated afterinoculation. From a statistical analysis of these data, levels ofresistance to Agrobacterium and crown gall formation are determined.Transformed grape plants that express a lytic peptide (e.g., Shiva-1 orcecropin B or both) having an increased level of resistance toAgrobacterium or crown gall disease or both relative to control plantsare taken as being useful in the invention.

By “increased level of resistance” is meant a greater level ofresistance or tolerance to a disease-causing pathogen or pest in atransgenic grapevine (or scion, rootstock, cell, or seed thereof) thanthe level of resistance or tolerance or both relative to a control plant(for example, a non-transgenic grapevine). In preferred embodiments, thelevel of resistance in a transgenic plant of the invention is at least5–10% (and preferably 30% or 40%) greater than the resistance of acontrol plant. In other preferred embodiments, the level of resistanceto a disease-causing pathogen is 50% greater, 60% greater, and morepreferably even 75% or 90% greater than a control plant; with up to 100%above the level of resistance as compared to a control plant being mostpreferred. The level of resistance or tolerance is measured usingconventional methods. For example, the level of resistance to a pathogenmay be determined by comparing physical features and characteristics(for example, plant height and weight, or by comparing disease symptoms,for example, delayed lesion development, reduced lesion size, leafwilting, shriveling, and curling, decay of fruit clusters, water-soakedspots, leaf scorching and marginal burning, and discoloration of cells)of transgenic grape plants.

In another working example, constitutive expression of a lytic peptide(e.g., Shiva-1 or cecropin B) is used to control the fungus Botrytis,the causative agent of bunch rot disease. Specifically, a plantexpression vector is constructed that contains a transgene sequence thatexpresses the lytic peptide(s). This expression vector is then used totransform somatic embryos according to the methods described above. Toassess resistance to fungal infection, transformed plants andappropriate controls are grown to approximately 30 cm vinelength, andyoung leaves and shoots are inoculated with a mycelial suspension ofBotrytis. For example, plugs of Botrytis mycelia are inoculated on eachside of the leaf midvein of developing leaves. Plants are subsequentlyincubated in a growth chamber at 30° C. with constant fluorescent lightand high humidity. Leaves of transformed and control grapevines are thenevaluated for resistance to Botrytis infection and disease according toconventional experimental methods. For this evaluation, for example, thenumber of lesions per leaf and percentage of leaf area infected arerecorded every twenty-four hours for seven days after inoculation. Fromthese data, levels of resistance to Botrytis are determined. Inaddition, if desired, fruit clusters can be sprayed with a suspension ofBotrytis and infection monitored at 15–20° C. at 90% relative humidityafter fifteen to twenty-four hours. Transformed grapevines that expressa lytic peptide gene having an increased level of resistance to Botrytisand infection and disease relative to control plants are taken as beinguseful in the invention.

Alternatively, to assess resistance at the whole plant level,transformed and control grapevines are transplanted to potting soilcontaining an inoculum of Botrytis. Plants are then evaluated forsymptoms of fungal infection (for example, wilting or decayed leaves)over a period of time lasting from several days to weeks. Again,transformed grapevines expressing the lytic peptide gene(s) having anincreased level of resistance to the fungal pathogen, Botrytis, relativeto control plants are taken as being useful in the invention.

OTHER EMBODIMENTS

The invention further includes analogs of any naturally-occurring lyticpeptide. Analogs can differ from the naturally-occurring lytic peptideby amino acid sequence differences, by post-translational modifications,or by both. In preferred embodiments, lytic peptide analogs used in theinvention will generally exhibit about 30%, more preferably 50%, andmost preferably 60% or even having 70%, 80%, or 90% identity with all orpart of a naturally-occurring lytic peptide amino acid sequence. Thelength of sequence comparison is at least 10 to 15 amino acid residues,preferably at least 25 amino acid residues, and more preferably morethan 35 amino acid residues. Modifications include chemicalderivatization of polypeptides, e.g., acetylation, carboxylation,phosphorylation, or glycosylation; such modifications may occur duringpolypeptide synthesis or processing or following treatment with isolatedmodifying enzymes. Lytic peptide analogs can also differ from thenaturally-occurring by alterations in primary sequence. These includegenetic variants, both natural and induced (for example, resulting fromrandom mutagenesis by irradiation or exposure to ethyl methylsulfate orby site-specific mutagenesis as described in Sambrook, Fritsch andManiatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press,1989, or Ausubel et al., supra). Also included are cyclized peptides,molecules, and analogs which contain residues other than L-amino acids,e.g., D-amino acids or non-naturally occurring or synthetic amino acids,e.g., β or γ amino acids.

In addition to full-length lytic peptides, the invention also includespeptide fragments. As used herein, the term “fragment,” means at least10 contiguous amino acids, preferably at least 15 contiguous aminoacids, more preferably at least 20 contiguous amino acids, and mostpreferably at least 30 to 40 or more contiguous amino acids. Fragmentsof lytic peptides can be generated by methods known to those skilled inthe art or may result from normal protein processing (e.g., removal ofamino acids from the nascent polypeptide that are not required forbiological activity or removal of amino acids by alternative mRNAsplicing or alternative protein processing events).

All publications mentioned in this specification are herein incorporatedby reference to the same extent as if each independent publication orpatent application was specifically and individually indicated to beincorporated by reference.

1. A method for increasing resistance to fungal disease in a grape plantcell, said method comprising transforming said grape plant cell with anucleic acid molecule which encodes a lytic peptide, wherein said lyticpeptide comprises the cecropin B peptide Shiva-1, wherein expression ofsaid peptide in said grape plant cell increases resistance of said grapeplant cell to fungal disease.
 2. The method of claim 1, furthercomprising propagating a grape plant from said plant cell.
 3. The methodof claim 1, wherein said plant cell is a scion cell.
 4. The method ofclaim 1, wherein said plant cell is a rootstock cell.
 5. The method ofclaim 1, wherein said plant cell is of the Thompson seedless grape. 6.The method of claim 1, wherein said fungal disease is bunch rot disease.7. A grape plant comprising a nucleic acid molecule which encodes alytic peptide, wherein said lytic peptide comprises the cecropin Bpeptide Shiva-1, wherein expression of said peptide in said grape plantincreases resistance of said grape plant to fungal disease.
 8. The plantof claim 7, wherein said nucleic acid molecule comprises an expressionvector.
 9. The plant of claim 7, wherein said plant is a scion cultivar.10. The plant of claim 7, wherein said plant is a rootstock cultivar.11. The plant of claim 7, wherein said plant is of the Thompson seedlessgrape.
 12. The plant of claim 7, wherein said fungal disease is bunchrot disease.
 13. A grape plant tissue wherein a cell of said tissuecomprises a nucleic acid molecule which encodes a lytic peptide, whereinsaid lytic peptide comprises the cecropin B peptide Shiva-1, whereinexpression of said peptide in said cell increases resistance of saidgrape plant tissue to fungal disease.
 14. The grape plant tissue ofclaim 13, wherein said plant tissue is a scion, a rootstock, a seed, ora somatic embryo.
 15. The grape plant tissue of claim 13, wherein saidnucleic acid molecule comprises an expression vector.
 16. The grapeplant tissue of claim 13, wherein said plant is of the Thompson seedlessgrape.
 17. The grape plant tissue of claim 13, wherein said fungaldisease is bunch rot disease.
 18. The method according to claim 6,wherein said bunch rot disease is caused by the fungus Botrytis.
 19. Themethod according to claim 12, wherein said bunch rot disease is causedby the fungus Botrytis.
 20. The method according to claim 17, whereinsaid bunch rot disease is caused by the fungus Botrytis.